JP6064039B2 - Method for detecting gene sensitive to low level ionizing radiation and gene detected by the method - Google Patents
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Description
本発明は、低レベル電離放射線に敏感な遺伝子の検出方法及び該方法により検出された遺伝子に係り、さらに詳しくは、癌誘発マウス及び正常マウスに低レベル放射線を照射して胸腺から正常マウス及び癌誘発マウスから共通して特異的に観察される糖代謝関連遺伝子を分類する低レベル電離放射線に敏感な遺伝子の検出方法に関する。
The present invention relates to a method for detecting a gene sensitive to low-level ionizing radiation and a gene detected by the method, and more particularly, to cancer-induced mice and normal mice by irradiating low-level radiation to normal mice and cancers from the thymus. The present invention relates to a method for detecting a gene sensitive to low-level ionizing radiation that classifies sugar metabolism-related genes that are specifically observed in common from induced mice.
産業及び医療的放射線の活用の増加とあいまって、人体影響への取り組みが盛んに行われており、主として放射線照射を用いた癌治療に関心が寄せられている。高線量電離放射線は、ＤＮＡ損傷、遺伝的変形、癌をはじめとする疾病を引き起こすが、２００ｍＧｙ以下の線量と６ｍＧｙ／時間以下の線量率放射線は免疫反応を活性化させて癌発生を抑制することが知られている。 Along with the increase in the utilization of industrial and medical radiation, efforts to influence the human body are being actively carried out, and there is an interest mainly in cancer treatment using radiation irradiation. High-dose ionizing radiation causes diseases such as DNA damage, genetic deformation, and cancer, but doses of 200 mGy or less and dose rate radiation of 6 mGy / hour or less activate the immune reaction and suppress cancer development. It has been known.
一般に、放射線と癌発生との間の相関性、特に、遺伝子反応への取り組みが行われてきたが、その結果の信頼性を阻害する交絡変数が多数介入されていた。しかしながら、これまで、ほとんどの研究では、遺伝子の一部を変形したり癌細胞株を用いたりしたため、身体を構成する細胞、組織、臓器、そして、身体段階で観察される様々な反応を説明することができなかった。すなわち、一般マウスを用いて遺伝子反応を評価したため、発現遺伝子が様々であっただけではなく、癌発生が特定の臓器に限定されていなかったため遺伝子反応を解析することが困難であったという問題がある。 In general, efforts have been made to correlate the correlation between radiation and cancer development, particularly genetic responses, but many confounding variables have been intervening that interfere with the reliability of the results. However, until now, most studies have modified parts of genes or used cancer cell lines to explain the various cells, tissues, organs, and various reactions observed at the body stage. I couldn't. That is, since gene reactions were evaluated using general mice, not only were the expressed genes varied, but there was a problem that it was difficult to analyze gene reactions because cancer development was not limited to specific organs. is there.
癌研究のために細胞を利用する従来の方法では、遺伝子を変更したり、癌発生で重要視されるｐ５３が欠如した癌細胞に放射線を照射したりしたため、正常細胞の反応とは根本的に異なる結果、その結果を個体に適用することができないという問題があり、このような欠点を補うために、ヒトと遺伝子が９５％以上同じマウスを用いて放射線に対する癌発生への取り組みを行っている。しかしながら、一般マウスは自然癌発生率が非常に低いため、様々な癌研究用モデルマウスが用いられている。 In conventional methods using cells for cancer research, genes are changed, or cancer cells lacking p53, which is important for cancer development, are irradiated with radiation. There is a problem that the results cannot be applied to individuals because of different results, and in order to make up for these drawbacks, efforts are being made to develop cancers against radiation using mice that are the same as humans by over 95%. . However, since general mice have a very low natural cancer incidence, various model mice for cancer research are used.
従来の研究では、低レベル（０．７ｍＧｙ／時間）放射線に敏感な糖代謝関連遺伝子を発掘するために種々の方法を利用した。しかしながら、本発明において提示した糖代謝関連遺伝子は、過去に低レベル（０．７ｍＧｙ／時間）放射線に対して敏感な遺伝子であることが知られていない。 Previous studies utilized various methods to uncover genes related to glucose metabolism that are sensitive to low levels (0.7 mGy / hour) of radiation. However, the sugar metabolism-related genes presented in the present invention have not been known in the past to be sensitive to low levels (0.7 mGy / hour) of radiation.
本願発明の低レベル（０．７ｍＧｙ／時間）放射線に敏感な糖代謝関連遺伝子プロファイルの発掘に先行された技術は、下記の通りである。 The technology prior to the discovery of the low-level (0.7 mGy / hour) radiation-sensitive glucose metabolism-related gene profile of the present invention is as follows.
（１）癌細胞は、糖吸収及び糖分解活性化の特徴を示している（Warburg O, Science 1956; 123: 309-314）。 (1) Cancer cells exhibit characteristics of sugar absorption and glycolysis activation (Warburg O, Science 1956; 123: 309-314).
（２）活性化された糖代謝は胸腺でｐ５３の活性を抑制し、ｐｕｍａの誘導を抑制し、Ｂｃｌ２ファミリタンパク質発現の細胞死滅抑制のバランスに影響を与え、癌生存を維持した（Zhao Y et. al., J Biol Chem 2008; 283: 36344-36353）。 (2) Activated glucose metabolism suppresses the activity of p53 in the thymus, suppresses the induction of puma, affects the balance of cell death suppression of Bcl2 family protein expression, and maintains cancer survival (Zhao Y et al. al., J Biol Chem 2008; 283: 36344-36353).
（３）電離放射線が照射され、且つ、Ａｋｔ１発現が抑制されたマウスの回腸で細胞死滅が増大された（Plastarasら, 2008）。 (3) Cell death was increased in the ileum of mice irradiated with ionizing radiation and suppressed in expression of Akt1 (Plastaras et al., 2008).
そこで、本発明者らは、低レベル電離放射線に対して敏感な糖代謝関連遺伝子プロファイル発掘することにより本発明を完成するに至った。
Accordingly, the present inventors have completed the present invention by finding a gene profile related to sugar metabolism that is sensitive to low-level ionizing radiation.
本発明の目的は、低レベル電離放射線に敏感な遺伝子の検出方法及び該方法により検出された遺伝子を提供するところにある。
An object of the present invention is to provide a method for detecting a gene sensitive to low-level ionizing radiation and a gene detected by the method.
前記目的を達成するために、本発明は、Ｉ）癌が誘発されたＡＫＲ／Ｊマウス及び正常のＩＣＲマウスに低レベル放射線を照射するステップと、II）前記ＡＫＲ／Ｊマウス及びＩＣＲマウスから胸腺を採取するステップと、III）前記胸腺に対してマイクロアレイを行うステップと、IV）前記マイクロアレイから糖代謝関連遺伝子を分類するステップと、Ｖ）前記遺伝子を増幅し、且つ、発現量を測定するステップと、を含み、癌誘発個体で発掘され、正常個体で検証される低レベル電離放射線に敏感な遺伝子の検出方法を提供する。 To achieve the above object, the present invention comprises: I) irradiating cancer-induced AKR / J mice and normal ICR mice with low level radiation; and II) thymus from the AKR / J mice and ICR mice. III) a step of microarraying the thymus, IV) a step of classifying a glucose metabolism-related gene from the microarray, and V) a step of amplifying the gene and measuring the expression level And a method for detecting a gene sensitive to low-level ionizing radiation that is excavated in a cancer-inducing individual and verified in a normal individual.
また、本発明は、胸腺癌悪化に関与する糖代謝関連遺伝子であるＩＲＳ１（ＮＭ＿０１０５７０）、Ｇｌｕｔ１（ＭＭ＿０１１４００）、Ｇｌｕｔ４（ＮＭ＿００９２０４）、ＬＰＫ（ＮＭ＿０１３６３１）及びＧ６ｐｃ（ＮＭ＿００８０６１）よりなる群から選ばれる遺伝子の塩基配列を含む放射線敏感性または放射線発癌診断用マーカーを提供する。 The present invention also relates to a gene selected from the group consisting of IRS1 (NM — 010570), Glut1 (MM — 011400), Glut4 (NM — 009204), LPK (NM — 013631) and G6pc (NM — 008061), which are sugar metabolism-related genes involved in thymic cancer deterioration. A marker for diagnosis of radiation sensitivity or radiation carcinogenesis including a base sequence is provided.
さらに、本発明は、上記のマーカーを備える放射線敏感性または放射線発癌診断用キットを提供する。 Furthermore, the present invention provides a radiation sensitive or radiation carcinogenic diagnosis kit comprising the above marker.
さらに、本発明は、上記のマーカーを備える放射線敏感性または放射線発癌診断用マイクロアレイを提供する。 Furthermore, the present invention provides a radiation sensitive or radiation carcinogenic diagnosis microarray comprising the above marker.
さらに、本発明は、Ｉ）ヒトを除く胸腺癌を有する哺乳動物に放射線を照射するステップと、II）前記放射線が照射された哺乳動物から摘出した胸腺組織と試験物質を接触させるステップと、III）前記胸腺組織から胸腺癌悪化に関与する糖代謝関連遺伝子であるＩＲＳ１（ＮＭ＿０１０５７０）、Ｇｌｕｔ１（ＭＭ＿０１１４００）、Ｇｌｕｔ４（ＮＭ＿００９２０４）、ＬＰＫ（ＮＭ＿０１３６３１）及びＧ６ｐｃ（ＮＭ＿００８０６１）よりなる群から選ばれる遺伝子の発現変化を測定するステップと、を含む放射線敏感性または放射線発癌を測定する遺伝子の検出方法を提供する。 Furthermore, the present invention includes I) a step of irradiating a mammal having thymic cancer excluding humans, II) a step of contacting a test substance with a thymus tissue extracted from the mammal irradiated with the radiation, and III ) Expression of a gene selected from the group consisting of IRS1 (NM — 010570), Glut1 (MM — 011400), Glut4 (NM — 009204), LPK (NM — 013631) and G6pc (NM — 008061), which are sugar metabolism related genes involved in thymic cancer deterioration from the thymic tissue Measuring a change, and a method for detecting a gene that measures radiation sensitivity or radiation carcinogenesis.
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
放射線に対する人体影響のうち癌発生への取り組みが盛んに行われてきたが、癌細胞若しくは遺伝子が変形された細胞株や一般マウスを用いたが故に様々な人体反応（遺伝子反応）を説明することが困難であった。特に、個体次元で電離放射線に敏感な糖代謝関連遺伝子プロファイルを発掘して機能を説明したことがない。この理由から、本発明では、１）正常的なＩＣＲマウスと胸腺癌を発病するＡＫＲ／Ｊマウスに低レベル（０．７ｍＧｙ／時間）放射線を照射（癌発生刺激因子）した後、胸腺で特異的に発現する低レベル放射線に敏感な糖代謝関連遺伝子プロファイルを発掘して機能を分析した後、２）胸腺癌発生段階を診断する糖代謝関連遺伝子プロファイルの構成因子として使用しようとした。 Although many efforts have been made to develop cancer among the effects of the human body on radiation, various human body reactions (genetic reactions) should be explained because cancer cells or cell lines with modified genes and general mice were used. It was difficult. In particular, we have not found a function by excavating a glucose metabolism-related gene profile that is sensitive to ionizing radiation in the individual dimension. For this reason, in the present invention, 1) normal ICR mice and AKR / J mice that develop thymic cancer are irradiated with low levels (0.7 mGy / hour) of radiation (cancer-stimulating factor) and then specific in the thymus. After digging out a low-level radiation-sensitive gene profile related to glucose metabolism and analyzing its function, 2) we attempted to use it as a component of the glucose metabolism-related gene profile for diagnosing the stage of thymic carcinoma development.
本発明は、Ｉ）癌が誘発されたＡＫＲ／Ｊマウス及び正常のＩＣＲマウスに低レベル放射線を照射するステップと、II）前記ＡＫＲ／Ｊマウス及びＩＣＲマウスから胸腺を採取するステップと、III）前記胸腺に対してマイクロアレイを行うステップと、IV）前記マイクロアレイから糖代謝関連遺伝子を分類するステップと、Ｖ）前記遺伝子を増幅し、且つ、発現量を測定するステップと、を含み、癌誘発個体で発掘され、正常個体で検証される低レベル電離放射線に敏感な遺伝子の検出方法を提供する。 The present invention comprises I) irradiating cancer-induced AKR / J mice and normal ICR mice with low level radiation, II) collecting thymus from said AKR / J mice and ICR mice, and III) Performing a microarray on the thymus, IV) classifying a glucose metabolism-related gene from the microarray, and V) amplifying the gene and measuring an expression level, comprising a cancer-inducing individual A method for detecting genes sensitive to low-level ionizing radiation excavated in and verified in normal individuals.
本発明の低レベル電離放射線に敏感な遺伝子の検出方法において、前記低レベル電離放射線は、ガンマ線（Ｃｓ−１３７）を０．７ｍＧｙ／時間の強さで最終的に１．７Ｇｙの放射線量で照射することが好ましく、本願発明の前記方法は、胸腺癌診断用キットの製作、癌患者の癌進行及び治療程度の評価、産業及び医療従事者の放射線被曝と発癌との間の相関性評価、放射線と癌発生との間の因果関係評価、放射線被曝に対する生物学的線量評価または低レベル放射線による胸腺癌発生及び進行度評価の指標として活用されることが好ましい。 In the method for detecting a gene sensitive to low-level ionizing radiation of the present invention, the low-level ionizing radiation is irradiated with gamma rays (Cs-137) at an intensity of 0.7 mGy / hour and finally with a radiation dose of 1.7 Gy. Preferably, the method of the present invention comprises a kit for diagnosing thymic cancer, evaluation of cancer progression and treatment level of cancer patients, evaluation of correlation between radiation exposure and carcinogenesis of industrial and medical personnel, radiation It is preferably used as an index for evaluating the causal relationship between cancer and cancer development, biological dose evaluation for radiation exposure, or evaluation of thymic cancer development and progression by low-level radiation.
また、本発明の低レベル電離放射線に敏感な遺伝子の検出方法において、前記癌は、胸腺癌であることが好ましく、前記ステップII）の胸腺の採取は、癌によって斃死が始まる時点で行われることが好ましい。 In the method for detecting a gene sensitive to low-level ionizing radiation according to the present invention, the cancer is preferably a thymic cancer, and the thymus collection in the step II) is performed at the time when moribund begins by the cancer. Is preferred.
さらに、本発明の低レベル電離放射線に敏感な遺伝子の検出方法において、前記糖代謝関連遺伝子は、ＩＲＳ１（ＮＭ＿０１０５７０）、Ｇｌｕｔ１（ＭＭ＿０１１４００）、Ｇｌｕｔ４（ＮＭ＿００９２０４）、ＬＰＫ（ＮＭ＿０１３６３１）及びＧ６ｐｃ（ＮＭ＿００８０６１）よりなる群から選ばれる遺伝子であることが好ましく、このとき、前記ＩＲＳ１（ＮＭ＿０１０５７０）遺伝子は配列番号１及び２に記載のプライマ、Ｇｌｕｔ１（ＭＭ＿０１１４００）遺伝子は配列番号３及び４に記載のプライマ、Ｇｌｕｔ４（ＮＭ＿００９２０４）遺伝子は配列番号５及び６に記載のプライマ、ＬＰＫ（ＮＭ＿０１３６３１）遺伝子は配列番号７及び８に記載のプライマ、且つ、Ｇ６ｐｃ（ＮＭ＿００８０６１）遺伝子は配列番号９及び１０に記載のプライマによって増幅されることが好ましい。 Furthermore, in the method for detecting a gene sensitive to low-level ionizing radiation of the present invention, the sugar metabolism-related gene is from IRS1 (NM — 010570), Glut1 (MM — 011400), Glut4 (NM — 009204), LPK (NM — 013631) and G6pc (NM — 008061). Preferably, the IRS1 (NM — 010570) gene is the primer described in SEQ ID NOs: 1 and 2, the Glut1 (MM — 011400) gene is the primer described in SEQ ID NOs: 3 and 4, and the Glut4 ( The NM_009204) gene is the primer described in SEQ ID NOs: 5 and 6, the LPK (NM_013631) gene is the primer described in SEQ ID NOs: 7 and 8, and the G6pc (NM_008061) gene is SEQ ID NO: 9 and It is preferably amplified by primers set forth in 0.
前記マイクロアレイから糖代謝関連遺伝子を分類する前記ステップIV）においては、放射線照射前の癌誘発個体に比べて放射線照射後の癌誘発個体で過剰発現または低発現された遺伝子をマイクロアレイを用いて検出した後、配列番号１番〜１０番のプライマを用いて検証し、遺伝子の機能検索法を用いて前記過剰発現または低発現された遺伝子の本質を究明することを特徴とする。マイクロアレイ分析の詳細については、下記の実施例において説明しており、遺伝子の機能検索は、下記の実施例において、生物情報データベースＤＡＶＩＤ（http://apps1.niaid.nih.gov/david/）及びＰｕｂｍｅｄデータベース（http://www.ncbi.nlm.nih.gov/）を用いて行ったが、これに限定されるものではない。 In the step IV) of classifying genes related to sugar metabolism from the microarray, genes that were overexpressed or underexpressed in the cancer-induced individual after irradiation compared to the cancer-induced individual before irradiation were detected using the microarray. Thereafter, verification is performed using primers of SEQ ID NOs: 1 to 10, and the essence of the overexpressed or underexpressed gene is investigated using a gene function search method. Details of the microarray analysis are described in the following examples, and gene function searches are performed in the following examples in the biological information database DAVID (http://apps1.niaid.nih.gov/david/) and Although it was performed using the Pubmed database (http://www.ncbi.nlm.nih.gov/), it is not limited to this.
本発明において使用する「低レベル電離放射線に敏感な遺伝子」とは、放射線を照射する前の癌誘発個体と比較して、放射線を照射した後の癌誘発個体で差別的に過剰発現または低発現される遺伝子のことをいう。すなわち、癌誘発個体で放射線刺激によって発現パターンの変化が誘導される遺伝子を意味し、特定の癌と関連したターゲット遺伝子、すなわち、腫瘍形成遺伝子または腫瘍抑制遺伝子であってもよい。このような癌特異的な遺伝子を検出することにより、癌患者に対する放射線治療の分子的な機序を確立して放射線治療効果の増大に寄与することができるだけではなく、新たな腫瘍形成または腫瘍抑制遺伝子を発掘してこれらの発現を調節することにより、分子生物学的レベルにおける癌治療剤または治療方法の開発のための基盤を設けることができる。 As used herein, “a gene sensitive to low-level ionizing radiation” is differentially overexpressed or underexpressed in a cancer-induced individual after irradiation as compared to a cancer-induced individual before irradiation. Refers to the gene That is, it means a gene whose expression pattern is changed by radiation stimulation in a cancer-induced individual, and may be a target gene associated with a specific cancer, that is, a tumorigenic gene or a tumor suppressor gene. By detecting such cancer-specific genes, not only can we establish a molecular mechanism of radiotherapy for cancer patients and contribute to the enhancement of radiotherapy effects, but also new tumor formation or tumor suppression. Excavating genes and regulating their expression can provide the basis for the development of cancer therapeutics or methods at the molecular biological level.
また、本発明は、胸腺癌悪化に関与する糖代謝関連遺伝子であるＩＲＳ１（ＮＭ＿０１０５７０）、Ｇｌｕｔ１（ＭＭ＿０１１４００）、Ｇｌｕｔ４（ＮＭ＿００９２０４）、ＬＰＫ（ＮＭ＿０１３６３１）及びＧ６ｐｃ（ＮＭ＿００８０６１）よりなる群から選ばれる遺伝子の塩基配列を含む放射線敏感性または放射線発癌診断用マーカーを提供する。 The present invention also relates to a gene selected from the group consisting of IRS1 (NM — 010570), Glut1 (MM — 011400), Glut4 (NM — 009204), LPK (NM — 013631) and G6pc (NM — 008061), which are sugar metabolism-related genes involved in thymic cancer deterioration. A marker for diagnosis of radiation sensitivity or radiation carcinogenesis including a base sequence is provided.
さらに、本発明は、上記のマーカーを備える放射線敏感性または放射線発癌診断用キットを提供する。 Furthermore, the present invention provides a radiation sensitive or radiation carcinogenic diagnosis kit comprising the above marker.
さらに、本発明は、上記のマーカーを備える放射線敏感性または放射線発癌診断用マイクロアレイを提供する。 Furthermore, the present invention provides a radiation sensitive or radiation carcinogenic diagnosis microarray comprising the above marker.
さらに、本発明は、Ｉ）ヒトを除く胸腺癌を有する哺乳動物に放射線を照射するステップと、II）前記放射線が照射された哺乳動物から摘出した胸腺組織と試験物質を接触させるステップと、III）前記胸腺組織から胸腺癌悪化に関与する糖代謝関連遺伝子であるＩＲＳ１（ＮＭ＿０１０５７０）、Ｇｌｕｔ１（ＭＭ＿０１１４００）、Ｇｌｕｔ４（ＮＭ＿００９２０４）、ＬＰＫ（ＮＭ＿０１３６３１）及びＧ６ｐｃ（ＮＭ＿００８０６１）よりなる群から選ばれる遺伝子の発現変化を測定するステップと、を含む放射線敏感性または放射線発癌を測定する遺伝子の検出方法を提供する。 Furthermore, the present invention includes I) a step of irradiating a mammal having thymic cancer excluding humans, II) a step of contacting a test substance with a thymus tissue extracted from the mammal irradiated with the radiation, and III ) Expression of a gene selected from the group consisting of IRS1 (NM — 010570), Glut1 (MM — 011400), Glut4 (NM — 009204), LPK (NM — 013631) and G6pc (NM — 008061), which are sugar metabolism related genes involved in thymic cancer deterioration from the thymic tissue Measuring a change, and a method for detecting a gene that measures radiation sensitivity or radiation carcinogenesis.
本発明では、胸腺癌研究モデルであるＡＫＲ／Ｊマウス及び正常的なマウス種であるＩＣＲに対して低レベル（０．７ｍＧｙ／時間）ガンマ線（Ｃｓ−１３７）を照射し、ＡＫＲ／Ｊマウスが胸腺癌によって斃死し始める時点（１００日目）を規定して胸腺を採取した。採取した胸腺に対してマイクロアレイを行った後にＤＡＶＩＤ生物情報データベースを用いて低レベル（０．７ｍＧｙ／時間）放射線に対して敏感な反応を示す糖代謝関連遺伝子を分類し、低レベル（０．７ｍＧｙ／時間）放射線に対して敏感な反応を示す糖代謝関連遺伝子を核酸増幅し、且つ、発現量を測定した。 In the present invention, a low level (0.7 mGy / hour) gamma ray (Cs-137) is irradiated to an ACR / J mouse that is a thymic cancer research model and an ICR that is a normal mouse species, and the AKR / J mouse The thymus was collected at a defined time (day 100) when dying due to thymic cancer began. Microarrays were performed on the collected thymus, and then the glucose metabolism-related genes showing a response sensitive to low level (0.7 mGy / hour) radiation were classified using the DAVID biological information database, and the low level (0.7 mGy / Time) Nucleic acid amplification of sugar metabolism-related genes showing a sensitive response to radiation, and the expression level was measured.
その結果、本発明により、低レベル（０．７ｍＧｙ／時間）放射線に対して敏感な反応を示す糖代謝（ＩＲＳ１、Ｇｌｕｔ１、Ｇｌｕｔ４、ＬＰＫ及びＧ６ｐｃ）に重要な５個の遺伝子を発掘し、低レベル（０．７ｍＧｙ／時間）放射線に対して敏感な反応を示す糖代謝（ＩＲＳ１、Ｇｌｕｔ１、Ｇｌｕｔ４、ＬＰＫ及びＧ６ｐｃ）関連遺伝子の機能を理解しやすいように提示した。なお、胸腺癌によって斃死が観察される１００日目を規定して胸腺を採取することにより、低レベル放射線に反応を示す糖代謝遺伝子反応を高い一貫性で観察できるようにした。 As a result, according to the present invention, five genes important for sugar metabolism (IRS1, Glut1, Glut4, LPK and G6pc) showing a response sensitive to low level (0.7 mGy / hour) radiation are excavated, and low Levels (0.7 mGy / hour) The functions of genes related to glucose metabolism (IRS1, Glut1, Glut4, LPK and G6pc) showing a sensitive response to radiation were presented for easy understanding. By collecting the thymus on the 100th day when moribund death is observed due to thymic cancer, it was possible to observe the glucose metabolism gene reaction that is responsive to low-level radiation with high consistency.
このため、本発明は、１）胸腺癌診断用診断キットの開発のための糖代謝関連遺伝子プロファイルとして活用することができ、２）低レベル放射線環境下で生活する産業及び医療従事者の癌発生因果関係評価の指標として活用することができ、３）癌患者の癌発生を診断し、且つ、治療対策を立てる情報用遺伝子プロファイルとして活用することができ、４）放射線被曝と胸腺癌発生との間の因果関係を区別する指標として活用することができ、５）低レベル放射線被曝に対する生物学的線量の評価のための新たな遺伝子指標として幅広く活用することができ、しかも、６）電離放射線に敏感な糖代謝シグナル伝達は、低レベル放射線被曝に対するターゲット治療に適用することができる。
Therefore, the present invention can be used as 1) a sugar metabolism-related gene profile for the development of a diagnostic kit for thymic cancer diagnosis, and 2) the occurrence of cancer in industrial and medical workers living in a low-level radiation environment. It can be used as an index for causal relationship evaluation, 3) can be used as an information gene profile for diagnosing cancer occurrence in cancer patients and making treatment measures, and 4) radiation exposure and thymic cancer occurrence 5) Can be widely used as a new genetic index for biological dose assessment for low-level radiation exposure, and 6) For ionizing radiation Sensitive glucose metabolism signaling can be applied to targeted therapies for low level radiation exposure.
上記の構成を有する低レベル電離放射線に敏感な遺伝子の検出方法は、胸腺癌診断用キットを製作するための低レベル（０．７ｍＧｙ／時間）放射線に敏感な糖代謝関連指標遺伝子プロファイルとして活用することができ、癌患者の癌進行及び治療程度を評価する低レベル（０．７ｍＧｙ／時間）放射線に敏感な糖代謝関連指標遺伝子として活用することができる。また、産業及び医療従事者の放射線被曝と発癌との間の相関性を評価する低レベル（０．７ｍＧｙ／時間）放射線に敏感な糖代謝関連指標遺伝子として活用することができ、放射線と癌発生との間の因果関係を評価する低レベル（０．７ｍＧｙ／時間）放射線に敏感な糖代謝関連指標として活用することができる。さらに、放射線被曝に対する生物学的線量の評価のための新たな指標として活用することができ、低レベル（０．７ｍＧｙ／時間）放射線による胸腺癌の発生及び進行度、抑制効果を評価する低レベル放射線に敏感な糖代謝関連指標遺伝子として活用することができる。
The method for detecting a gene sensitive to low-level ionizing radiation having the above-described configuration is utilized as a low-level (0.7 mGy / hour) radiation-sensitive glucose metabolism-related indicator gene profile for producing a thymic cancer diagnostic kit. It can be used as a low-level (0.7 mGy / hour) radiation sensitive glucose metabolism-related indicator gene to evaluate cancer progression and the extent of treatment of cancer patients. It can also be used as a low-level (0.7 mGy / hour) radiation-sensitive glucose metabolism-related indicator gene to evaluate the correlation between radiation exposure and carcinogenesis of industrial and medical workers. It can be used as a low-level (0.7 mGy / hour) radiation-sensitive glucose metabolism-related index for evaluating the causal relationship between the two. Furthermore, it can be used as a new index for the evaluation of biological dose for radiation exposure, and it is a low level to evaluate the occurrence and progression of thymic cancer by low level (0.7 mGy / hour) radiation, and the inhibitory effect. It can be used as an indicator gene related to sugar metabolism sensitive to radiation.
以下、本発明を実施例を挙げてより具体的に説明する。しかしながら、下記の実施例は本発明を説明するためのものに過ぎず、下記の実施例によって本発明の権利範囲が限定されることはない。
Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples are only for explaining the present invention, and the scope of rights of the present invention is not limited by the following examples.
＜実施例１＞
日本ＳＬＣ社から６週齢の胸腺癌研究モデルである雌性ＡＫＲ／Ｊマウス及び正常の雌性ＩＣＲマウスを購入した。ガンマ線発生装置（ＩＢＬ １４７Ｃ、ＣＩＳ ｂｉｏ ｉｎｔｅｒｎａｔｉｏｎａｌ、Ｆｒａｎｃｅ）を用いて前記ＡＫＲ／Ｊマウスに低レベル放射線を最終線量が４．５Ｇｙに達するように照射（１３７Ｃｓ、０．７ｍＧｙ／時間）した。低レベル放射線を照射し終わったマウスは放射線が遮断された無菌飼育施設に移して１００日間飼育しながら胸腺癌の発生を観察した。遺伝子分析のために同じ実験条件下で別途に飼育した正常マウス（ＩＣＲマウス）に低レベル（０．７ｍＧｙ／時間）放射線を照射し、１００日後に採取した胸腺は液体窒素を用いて急速凍結させた後に遺伝子分析を行った。
<Example 1>
Female AKR / J mice and normal female ICR mice, which are 6-week-old thymic cancer research models, were purchased from Japan SLC. Using a gamma ray generator (IBL 147C, CIS bio international, France), the AKR / J mice were irradiated with low-level radiation to reach a final dose of 4.5 Gy ( 137 Cs, 0.7 mGy / hour). Mice that had been irradiated with low-level radiation were transferred to a sterile breeding facility where radiation was blocked and bred for 100 days to observe the occurrence of thymic carcinoma. Normal mice (ICR mice) reared separately under the same experimental conditions for genetic analysis were irradiated with low level (0.7 mGy / hour) radiation, and the thymus collected 100 days later was rapidly frozen using liquid nitrogen. After that, gene analysis was performed.
＜実施例２＞ マイクロアレイ及び遺伝子分析
前記実施例１の結果、癌研究用モデルマウス（ＡＫＲ／Ｊマウス）を用いて低レベル（０．７ｍＧｙ／時間）放射線に敏感な糖代謝関連遺伝子を発掘し、これを正常マウス（ＩＣＲマウス）で証明した。すなわち、低レベル（０．７ｍＧｙ／時間）放射線が照射されたＡＫＲ／Ｊマウス及びＩＣＲマウスの胸腺で特異的に低レベル放射線に反応する糖代謝関連遺伝子を発掘し、且つ、機能を解析した。ＤＡＶＩＤ生物情報データベース、定量的核酸増幅法及び統計プログラムＳＡＳ（ＡＮＯＶＡとｔ−ｔｅｓｔ）を用いて分析した。
<Example 2> Microarray and gene analysis As a result of the above-mentioned Example 1, a sugar metabolism-related gene sensitive to low level (0.7 mGy / hour) radiation was found using a model mouse for cancer research (AKR / J mouse). This was verified with normal mice (ICR mice). That is, a sugar metabolism-related gene that specifically reacts with low-level radiation was excavated and analyzed in the thymus of AKR / J mice and ICR mice irradiated with low-level (0.7 mGy / hour) radiation. Analysis was performed using the DAVID bioinformation database, quantitative nucleic acid amplification method and statistical program SAS (ANOVA and t-test).
その結果を確認するために、前記遺伝子を核酸増幅した。具体的に、低レベル（０．７ｍＧｙ／時間）放射線が照射されたＡＫＲ／Ｊマウス及びＩＣＲマウスから採取した胸腺に対してマイクロアレイを行い、低レベル放射線に対して敏感な反応を示す糖代謝関連遺伝子に対して発現量を計測するために下記表１に記載のプライマを用いて増幅した。 In order to confirm the result, the gene was amplified by nucleic acid. Specifically, microarray was performed on thymus collected from AKR / J mice and ICR mice irradiated with low level (0.7 mGy / hour) radiation, and glucose metabolism-related showed a sensitive response to low level radiation In order to measure the expression level for the gene, amplification was performed using the primers described in Table 1 below.
また、ＡＫＲ／Ｊマウス及びＩＣＲマウスに低レベル（０．７ｍＧｙ／時間）放射線を照射した後に飼育しながら、ＡＫＲ／Ｊマウスが胸腺癌で斃死し始める時点（１００日目）で胸腺を採取した。採取した胸腺に対してマイクロアレイを行い、低レベル放射線に対して敏感な反応を示す糖代謝関連遺伝子を分類した後、核酸を増幅して発現量を測定した。その結果、低レベル放射線が照射されたマウスでは、糖代謝関連遺伝子（ＩＲＳ１、Ｇｌｕｔ１、Ｇｌｕｔ４、ＬＰＫ及びＧ６ｐｃ）が低レベル放射線に敏感に反応した。その結果を下記表２に示す。 In addition, the thymus was collected at the time when the AKR / J mice began to die from thymic cancer (day 100) while being reared after irradiating AKR / J mice and ICR mice with low level (0.7 mGy / hour) radiation. . The collected thymus was microarrayed to classify sugar metabolism-related genes showing a sensitive response to low-level radiation, and then the nucleic acid was amplified and the expression level was measured. As a result, in mice irradiated with low-level radiation, sugar metabolism-related genes (IRS1, Glut1, Glut4, LPK and G6pc) responded sensitively to low-level radiation. The results are shown in Table 2 below.
また、図１に、低レベル（０．７ｍＧｙ／時間）放射線を照射して胸腺癌を抑制する糖代謝関連遺伝子の機能を理解しやすいように図式化して示す。低レベル放射線の照射はインスリンシグナル伝達（ＩＲＳ１）、糖吸収（Ｇｌｕｔ１及びＧｌｕｔ４）、糖分解（ＬＰＫ）及び新糖合成（Ｇ６ｐｃ）遺伝子の発現を低減し、胸腺癌を抑制した。 In addition, FIG. 1 is a schematic diagram for easy understanding of the function of a sugar metabolism-related gene that suppresses thymic cancer by irradiation with low-level (0.7 mGy / hour) radiation. Low level radiation reduced the expression of insulin signaling (IRS1), sugar absorption (Glut1 and Glut4), glycolysis (LPK) and new sugar synthesis (G6pc) genes and suppressed thymic carcinoma.
さらに、図２は、ＡＫＲ／Ｊマウス及びＩＣＲマウスに低レベル（０．７ｍＧｙ／時間）放射線を照射した後に飼育しながら、ＡＫＲ／Ｊマウスが胸腺癌で斃死し始める時点（１００日目）を規定して胸腺を採取し、胸腺の重さを計測し、これを指標として放射線に敏感な糖代謝関連遺伝子反応を分析した。本発明により、胸腺癌によって斃死が発生する癌発病の初期段階を規定して胸腺を採取し、重さを比較することにより、低レベル放射線に対して敏感な反応を示す糖代謝関連遺伝子を常に高い一貫性で測定することが可能になった。 Further, FIG. 2 shows the time point (day 100) when AKR / J mice began to die from thymic cancer while being reared after irradiating AKR / J mice and ICR mice with low level (0.7 mGy / hour) radiation. The thymus was collected by stipulation, the thymus was weighed, and this was used as an index to analyze radiation-sensitive glucose metabolism-related gene reactions. According to the present invention, the thymus is collected by defining the initial stage of cancer onset that causes moribundity by thymic cancer, and by comparing the weight, a glucose metabolism-related gene that shows a sensitive response to low-level radiation is always detected. It became possible to measure with high consistency.
Claims (7)
II）前記ＡＫＲ／Ｊマウス及びＩＣＲマウスから胸腺を採取するステップと、
III）前記胸腺に対してマイクロアレイを行うステップと、
IV）前記マイクロアレイから、配列番号１１に記載のＩＲＳ１（ＮＭ＿０１０５７０）、配列番号１２に記載のＧｌｕｔ１（ＭＭ＿０１１４００）、配列番号１３に記載のＧｌｕｔ４（ＮＭ＿００９２０４）、配列番号１４に記載のＬＰＫ（ＮＭ＿０１３６３１）及び配列番号１５に記載のＧ６ｐｃ（ＮＭ＿００８０６１）よりなる群から選ばれる糖代謝関連遺伝子を分類するステップと、
Ｖ）前記遺伝子を増幅し、且つ、発現量を測定するステップと、
を含み、
癌誘発個体で発掘され、正常個体で検証される低レベル電離放射線に敏感な遺伝子の検出方法。 In AKR / J mice and normal ICR mice I) thymic carcinoma was induced, finally irradiated with a radiation dose of 1.7Gy as low levels of radiation gamma rays (Cs-137) with the intensity of 0.7MGy / Time And steps to
II) collecting thymus from said AKR / J mouse and ICR mouse;
III) performing a microarray on the thymus,
IV) From the microarray , IRS1 (NM — 010570) described in SEQ ID NO: 11, Glut1 (MM — 011400) described in SEQ ID NO: 12, Glut4 (NM — 009204) described in SEQ ID NO: 13, LPK (NM — 036331) described in SEQ ID NO: 14, and Classifying a glucose metabolism-related gene selected from the group consisting of G6pc (NM_008061) described in SEQ ID NO: 15 ;
V) amplifying the gene and measuring the expression level;
Including
A method of detecting genes sensitive to low-level ionizing radiation that is excavated in cancer-induced individuals and verified in normal individuals.
II）前記放射線が照射された哺乳動物から摘出した胸腺組織と試験物質を接触させるステップと、
III）前記胸腺組織から胸腺癌悪化に関与する糖代謝関連遺伝子である配列番号１１に記載のＩＲＳ１（ＮＭ＿０１０５７０）、配列番号１２に記載のＧｌｕｔ１（ＭＭ＿０１１４００）、配列番号１３に記載のＧｌｕｔ４（ＮＭ＿００９２０４）、配列番号１４に記載のＬＰＫ（ＮＭ＿０１３６３１）及び配列番号１５に記載のＧ６ｐｃ（ＮＭ＿００８０６１）よりなる群から選ばれる遺伝子の発現変化を測定するステップと、
を含む放射線敏感性または放射線発癌を測定する遺伝子の検出方法。 I) irradiating a mammal with thymic cancer except humans with radiation;
II) contacting the test substance with a thymus tissue extracted from the mammal irradiated with the radiation;
III) IRS1 (NM — 010570) described in SEQ ID NO: 11, Glut1 (MM — 011400) described in SEQ ID NO: 12, Glut4 (NM — 009204) described in SEQ ID NO: 13, which is a sugar metabolism-related gene involved in thymic cancer deterioration from the thymic tissue Measuring the change in expression of a gene selected from the group consisting of LPK (NM — 03631) described in SEQ ID NO: 14 and G6pc (NM — 008061) described in SEQ ID NO: 15 ;
A gene detection method for measuring radiation sensitivity or radiation carcinogenesis.
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